Myosin-interacting guanine exchange factor (MyoGEF) regulates the invasion activity of MDA-MB-231 breast cancer cells through activation of RhoA and RhoC

Abstract

The small guanine triphosphatase (GTPase) proteins RhoA and RhoC are essential for tumor invasion and/or metastasis in breast carcinomas. However, it is poorly understood how RhoA and RhoC are activated in breast cancer cells. Here we describe the role of myosin-interacting guanine nucleotide exchange factor (Myo-GEF) in regulating RhoA and RhoC activation as well as cell polarity and invasion in an invasive breast cancer cell line MDA-MB-231. RNA-interference (RNAi)-mediated depletion of MyoGEF in MDA-MB-231 cells not only suppresses the activation of RhoA and RhoC, but also decreases cell polarity and invasion activity. The dominant-negative mutants of RhoA and RhoC, but not Rac1 and Cdc42, dramatically decrease actin polymerization induced by MyoGEF. In addition, MyoGEF co-localizes with nonmuscle myosin IIA (NMIIA) to the front of migrating cells, and depletion of NMIIA by RNAi disrupts the polarized localization of MyoGEF at the cell leading edge, suggesting a role for NMIIA in regulating MyoGEF localization and function. Moreover, MyoGEFprotein levels significantly increase in infiltrating ductal carcinomas as well as in invasive breast cancer cell lines. Taken together, our results suggest that MyoGEF cooperates with NMIIA to regulate the polarity and invasion activity of breast cancer cells through activation of RhoA and RhoC.

Figure 1. MyoGEF is required for the invasion activity of MDA-MB-231 cells

(A) Immunoblot analysis with anti-MyoGEF antibody shows that MyoGEF is expressed in MDA-MB-231 and MDA-MB-435S cells, but not in MDA-MB-361, MDA-MB-468, and MCF-7 cells. (B) Immunoblot analysis confirms the depletion of MyoGEF in MDA-MB-231 cells by RNAi. (C) MDA-MB-231 cells depleted of MyoGEF and/or NMIIA were subjected to Matrigel invasion assays. (D) Images in (C) were quantitated by using the NIH ImageJ program. (E) Immunohistochemical analysis of a breast cancer tissue array with MyoGEF antibody. Three arrays were analyzed independently and similar results were obtained. Immunohistochemistry with preimmune serum shows light, background straining (data not shown). Images in (C) and (E) were taken by using a 20x objective (Leica DMI 6000 B microscope).

Figure 2. Depletion of MyoGEF represses RhoA and RhoC activation in MDA-MB-231 cells

(A) Immunoblot analysis confirms the depletion of MyoGEF in MDA-MB-231 cells by RNAi. (B) The image in (A) was quantitated by using the NIH ImageJ program to estimate the efficiency of MyoGEF depletion in MDA-MB-231 cells by RNAi. (C-F) Depletion of MyoGEF decreases the amount of active RhoA (C) and RhoC (D), but not Rac1 (E) and CDc42 (F), in MDA-MB-231 cells. ~6% of transfected cell lysates were used as control to estimate the amount of total RhoA, RhoC, Rac1, and Cdc42. (G) The images in (C), (D), (E), and (F) were quantitated by using the NIH ImageJ program.

Figure 3. In vitro activation of RhoA, RhoC, and Rac1, but not Cdc42, by MyoGEF

(A-D) The immunoprecipitated Myc-MyoGEF from transfected HeLa cells could activate RhoA (A), RhoC( B), and Rac1 (C), but not Cdc42 (D) in a fluorescence-based GEF assay. (E) ThioHis-MyoGEF (full-length) could bind both GDP-RhoA (lane 4) and GTP-RhoA (lane 5). (F) A MyoGEF fragment (amino acids 71-388) that contain the DH domain could bind both GDP-RhoC (lane 4) and GTP-RhoC (lane 5). (G) ThioHis-MyoGEF could bind GTP-Rac1 (lane 5) but not GDP-Rac1 (lane 4). D, preloaded with GDP; T, preloaded with GTP.

Figure 4. MyoGEF colocalizes with actin-myosin filaments at the cell leading edge

(A) MDA-MB-231 cells were subjected to immunofluorescence with anti-MyoGEF antibody (green) and rhodaminephalloidin (red). (B) Immunoblot analysis of total cell lysates from MDA-MB-231 with anti-MyoGEF antibody. Note that a single band was recognized by MyoGEF antibody in MDA-MB-231 cell lysates. (C) Exogenously expressed GFP-MyoGEF (green) colocalizes with actin filaments (red) in transfected MDA-MB-231 cells. (D) Exogenously expressed GFP-IIA (green) colocalizes with endogenous MyoGEF (red) at the cell leading edge of transfected MDA-MB-231 cells. (E) Exogenously expressed GFP-MyoGEF (green) colocalizes with endogenous NMIIA (red) at the cell leading edge of transfected MDA-MB-231 cells. Bars, 10 μm.

Figure 5. MyoGEF interacts with NMIIA

(A) MDA-MB-231 cells expressing Myc-MyoGEF were subjected to immunoprecipitation with anti-Myc antibody followed by immunoblot analysis with anti-IIA or anti-IIB antibodies. Note that Myc-MyoGEF binds to NMIIA but not NMIIB. (B) Schematic diagram of MyoGEF fragments that were used in (C). (C) Interactions between Myc-tagged MyoGEF fragments and endogenous NMIIA. Full-length MyoGEF (lane 3) as well as MyoGEF fragments Myc-PH (lane 5), Myc-1-409 (lane 8), and Myc-1-500 (lane 9) could pull down a significant amount of endogenous NMIIA. Note that cell lysate from lane 3 was also used for immunoprecipitation with normal IgG (lane 2). ~5% of cell lysates were loaded.

Figure 6. Depletion of MyoGEF by RNAi impairs MDA-MB-231 cell polarity

(A) MDA-MB-231 cells treated with control siRNA (siCont) or MyoGEF siRNA (siMyoGEF) for 48 h were trypsinized, replated on fibronectin-coated coverslips, and cultured for an additional 6 h. Note that cells depleted of MyoGEF did not polarize. (B) Quantitation of nonpolarized MDA-MB-231 cells treated with control or MyoGEF siRNAs. (C) MDA-MB-231 cells treated with siCont or siMyoGEF were subjected to immunofluorescence with MyoGEF antibody (red) and FITC-phalloidin (green). (D) MDA-MB-231 cells treated with siCont or siMyoGEF were stained with antibodies specific for p-MRLC (green) and NMIIA (red). (E) MDA-MB-231 cells treated with siCont or siMyoGEF were stained with antibodies specific for p-MRLC (green) and NMIIB (red). Bar in (A), 80 μm; Bars in (C), (D), and (E), 10 μm

Figure 7. NMIIA is required for polarized localization of MyoGEF as well as the formation of MyoGEF-induced actin bundles

(A) MDA-MB-231 cells treated with control siRNA (siCont; panel a) or NMIIA siRNA (siIIA; panel b) were subjected to immunofluorescence with anti-MyoGEF antibody. (B) MDA-MB-231 cells treated with siCont or siIIA were subjected to immunoblot analysis with antibodies specific for NMIIA or β-tubulin. (C-D) A plasmid encoding GFP-MyoGEF was cotransfected into HeLa cells with siCont or siIIA. The transfected cells were subjected to immunofluorescence with anti-IIA antibody (C) or phalloidin (D). Bars, 10 μm.

Figure 8. Expression of dominant negative mutants N19RhoA and N19RhoC inhibits the formation of MyoGEF-induced actin bundles

A plasmid encoding GFP-MyoGEF was cotransfected into HeLa cells with an empty vector (a-c) or plasmids encoding N19RhoA (d-f), N19RhoC (g-i), N17Rac1 (j-l), or N17Cdc42 (m-o). Note that co-transfection of N19RhoA or N19RhoC decreases the formation of massive actin bundles induced by GFP-MyoGEF. Bars, 60 μm.